JPH07502598A - Devices for direct swab automated electrophoresis, transfer and detection, and methods for practical implementation of the devices - Google Patents

Abstract

A separation and direct blot transfer module useful in gel electrophoresis is disclosed in which a transfer membrane stabilized by a frame is moved substantially orthogonally relative to the plane of the separation gel while in minimal contact with the end of the gel toward which molecular fragments are driven electrophoretically. The frame moves at a controlled velocity in a selected direction away from a line of contact that generates a controlled tension in the membrane which is a function of the loading on the line of contact. The module is particularly amenable to automated processing. Apparatus for automated separation, transfer, membrane processing, detection and identification is disclosed in which the module of the invention is employed.

Description

[Detailed description of the invention] name of invention Devices for direct swab automated electrophoresis, transfer and detection, and practical use of the devices method field of invention The present invention relates to the field of gel electrophoresis. More specifically, the present invention Electrophoresis is carried out across the membrane to transfer the electrophoretically driven molecular components to the membrane. on a device for the precise separation above, and on how to use the above device for this purpose. It is something.

Background of the invention Gel electrophoresis is an important tool in analytical and clinical laboratories. gel electric Aerophoresis uses the electrodynamic differences of ions in an electric field to achieve separation of complex mixtures. It is used to identify or isolate each component. Agarose Gels such as polyacrylamide or polyacrylamide are most commonly cast into molds. A relatively thin rectangular cross-section expansion block is manufactured. A depression is formed at one end. child Support the block and immerse it in a buffer solution in a device with electrodes at each end. This hollow The gel is then loaded with a solution containing the substance under study, for example a nucleic acid, and An electric field is applied with a large gradient along the direction. Each molecule has dimensions and charge density It moves towards different electrodes at different speeds depending on its mobility which is a function. this There are many variations of the methodology.Gels have a continuous or stepwise density gradient. voltage or temperature may be controlled, e.g. by controlled polarity reversal. can be changed or changed; also pH gradients or concentration gradients You can also make

Two basic modes of operation are used, regardless of the particular arrangement used. usually In this method, separation is performed for a finite time, and then the pattern of the separated molecules is read. Certain known techniques, such as staining or radioactive or fluorescent It can be read on the spot by labeling using substances. Instead of this, various known techniques can be used. techniques, such as Southern wiping, pressure wiping, vacuum wiping or electric wiping (transverse electric field), etc., to transport the pattern in the orthogonal direction. separate Selective staining, radioactive labeling, fluorescent and chemical Immobilization on a membrane such as nylon allows the molecule to be identified by any method such as luminescence. Ru. This method uses a suitable probe to attach a labeling means to the separated chemical species. You can also

Other modalities often used in manufacturing electrophoresis involve coupled gel separation. continuously (or over an extended period of time) to transport the separated species from the end of the gel in a fixation cycle. Move to storage room. Occasionally, the contents of this chamber are emptied in a controlled manner to allow the departure mix. A stepwise separation of each component of the compound is obtained.

U.S. Patent 4.631.120 and 4.631.422 t: Paul ( Pohl) presented an apparatus and method for a variation of the latter method called the direct wiping method. is suggesting. One or more poles placed on a conveyor belt or tape The belt is advanced to slide over the end of the gel, providing a collecting surface of The particles are collected and removed from the gel using a belt for further processing, e.g. identification. He is being transported to a place where he will be humiliated. Both vertical and horizontal examples are disclosed. Ru. Paper by Beck and Paul (1'ohl) (EMROJ), 3 (12), 2905.1984) , the authors found that in this type of system, the belt for the r-axis (i.e., This emphasizes that the close contact between the belt and the gel end is achieved by . The method and apparatus are described as being particularly suitable for automation. .

The disadvantage of Paul's device is that it is supported by rollers only at relatively distant contact lines. Inherent to the use of tapes or belts is the plane stability and tension control of the collection plane. It is the lack of. Control of contact pressure is poor. In addition, the contact of the roller on the collection plane Touching may occur due to surface damage and may also result in the formation of certain separated chemical species. Disadvantages due to removal and subsequent reimprinting of the removed material in the wrong position on the surface Will.

Furthermore, specifically the separated elements are transferred to the membrane and these are subsequently separated into several different If the material must be transported through the processing stages for identification etc. The device is not acceptable for sequential multiple sample handling and identification.

Erlinger's U, S, 3.948.753 are different consisting of a capillary tube communicating with the sampling zone of the sampling column between the electrolytes at a potential of An apparatus for isotaphoretic separation is disclosed.

Everaerts U, S, 3,705.845 are countercurrent constant velocity Discloses a method in electrophoresis. A column with two electrodes moves differently A sample containing ions to be separated having a specific property is held. From each sample ion Introducing first and second electrolytes with high mobility and low mobility.

After applying voltage and pressure, the boundaries between ion bands are detected and a control signal is generated. let

Raymond's U, S, 3.047.489 is band electricity It is aimed at electrophoresis equipment. This patent describes a preferred connecting buffer membrane chamber, an attached electrode, A cold plate and various buffers and wicks are disclosed. U, S of Raymond et al. , 3.129.158 is used to pass an electric current through a gel and to add a sample to it. It includes means for producing a gel of the preferred size, shape and location for applying the mixture. Discloses a gel electrophoresis method comprising:

Durum (Durru+o) U, S, 3.062.731 is electrophoretic analysis Discloses agar systems and additives suitable for.

U.S. 4,622.124 Horizontal electric wiping of electrophoretically transferred substances It is aimed at equipment for use in the field. a support placed horizontally within the chamber defined by the container A liquid tight container having a set is provided. Electrodes are placed above and below this support, A foam barrier is provided to prevent uneven wiping.

U, S, 4.589.965 were electrophoretically resolved within the gelatin notebook. An electric wiping method that transfers materials to a membrane with high patterning and resolution capabilities. Disclosed. This gelatin sheet is sandwiched between two plate electrodes. contact with the immobilizing material of the combination. Apply a current to this set of electrodes.

U, S, 3.674.678 is a container filled with conductive gel, inside this container an electrophoretic analysis comprising two electrodes and means for applying a voltage across the gel. Discloses an apparatus for carrying out. This container has deep reservoirs on opposite ends. and a central recess for receiving the sample.

Transfer membrane to electrophoretic gel so that electrophoretic transfer to the membrane occurs in a controlled manner It is an aim of the present invention to provide an electrophoresis device that can be placed in the opposite direction. When visualized This transfer gives rise to electrophoretic patterns that are strikingly clear and reproducible. This is another objective of the present invention. Aspects of the invention provide that the transfer membrane is an electrophoretic gel. means for applying a controlled tension to the membrane when in contact with the membrane. This matter that the apparatus disclosed in the specification is suitable for automated processes involving various processing locations; is an advantage of the present invention. These and other objectives, aspects and benefits of the invention The points will become clear with reference to the description below.

Summary of the invention The specification includes: (a) an electrophoresis module containing a gel containing a solution with molecular fractions; Means of performing electrophoresis along this: (b) along the periphery of the flexible membrane; An electrophoretic transfer membrane comprising a rigid frame supporting a membrane of: (C) Relative to the above electrophoresis module, the electrophoresis transport membrane is The molecular fraction is transferred from the gel to the flexible membrane by contacting and moving the membrane. and (d) said flexible membrane is attached to said electrophoresis module. in solution, including means for tensioning this flexible membrane as it moves into close proximity. An apparatus for gel electrophoresis that electrophoretically separates molecular fractions across a gel is disclosed. ing.

In the electrophoresis module (a), the means for performing electrophoresis are (]) a cassette having a first reservoir adapted to receive a buffer and a first electrode; said first reservoir is adapted to receive said first reservoir; and placed in the first position. This module also (11) a buffer solution and a second electrode positioned in a second position with respect to the extended portion; It also includes a second storage tank. The gel containing the solution with molecular fractionation is the extended section above. placed between the first storage tank and the second storage tank, the first electrode The molecular fractionation is carried out by applying a potential difference between the electrophoretic transport membrane (b) and the second electrode. Electrophoretically transport and separate towards one end of the above gel in close proximity to.

The specification also includes (a) Electrophoresis along a gel containing a solution with a molecular fraction within it. and into an electrophoresis module having means for performing the motion.

(b) an electrophoretic transfer membrane comprising a rigid frame supporting a flexible membrane along its periphery; in contact with the above gel.

(c), J: Relative of the electrophoretic transport membrane described above to the electrophoretic module described above. The molecular fraction is then transferred from the gel onto the flexible membrane. Ketori 1 (d) the electrophoretic transport membrane moves in close proximity to the electrophoretic module; Gel electrophoresis of molecular fractions, including each step of applying tension to the flexible membrane described above. A method is also disclosed.

Brief description of the drawing FIG. 1 is an elevational view, partially in section, of a device according to the invention.

FIG. 2 is a top view of the device according to the invention.

Figure 3 shows a cross-section of the part of the device that holds the gel cassette, which is inserted into the gel cassette. FIG. 6 is an elevational view showing a gel with a preferred fully filled membrane-buffer supply. It shows the part.

FIG. 3A shows the membrane-buffer slot and wick supply in cross-section of the device of FIG. FIG.

FIG. 4 is an enlarged elevational view in cross-section of the membrane-buffer supply region for the preferred apparatus of FIG. It is.

Figure 4A shows the attached structure in the cassette of Figure 3A using slots and wicks. FIG. 2 is an enlarged elevation view in cross section.

FIG. 4B is similar to FIG. 4A, but shows a specific example in which a slot feeder is used instead of a core. It shows.

FIG. 5 is a general view of a frame holding a transfer membrane according to the invention.

Figure 6 shows the present invention incorporating automated fabrication of the transfer membrane to provide identification of separated species. 1 is a schematic overview of a preferred embodiment; FIG.

FIG. 7 is a general view of an embodiment of the apparatus of FIG. 1 configured for bench separation operations.

FIG. 8 is an overall view of the vertical transfer mechanism used in the example of FIG.

FIG. 9 is a cross-sectional elevational view of a pressure relief mechanism used in the load of the present invention using a transfer membrane. Ru.

FIG. 10 is an elevational view, partially in section, of a preferred simplified embodiment with loading means; .

detailed description of the invention The apparatus according to the invention shown in FIGS. 1 and 2 is generally designated as 10. base 12 is placed on the level table 14. Bio-Rad ) of level tables such as catalog number 170-4046 sold by Use or preferred. The support frame 16 is tightened on the base 12 by a tightening tool (not shown). and this support frame is tightened thereto by means, also not shown. The stepping motor 18 is held in place. Binion 20 is motor 18 The rack is installed on the shaft of the support frame 1 and engages with the rack 22. A guide 24 which is tightened at 6 and also functions to maintain proper gear meshing. Therefore, it is constrained to vertical sliding motion. This device collectively consists of a motor and drive a linkage set, which moves the electrophoretic transfer membrane relative to the electrophoretic module. is used for. Rack 22 is part of membrane carrier 26. Figure 5 best The electrophoretic transport membrane seen supports membrane 30 and is supported by means not shown. It is clamped to the carrier 26 along its upper edge by the clamp 32 which is tightened. It includes the frame 28 that is

Horizontal electrophoresis module 34 is on top of base 12 and fits into cavity 150. It is. This module 34 has a gel 42 cast therein and a gel 42 for holding it. It includes a cassette 44 and a slidable set 70. The cassette 44 A first reservoir t is located at one end with a lateral wire electrode 38 mounted on a rotating arm 140. 'l　36. The preferred constituent material for this electrode is that only the base of the wire is Platinum-plated titanium wire held in exposed molded insulation. Ru. Arm 140 is rotatably attached to base 12.

As seen in the figure, the gel transport occurs in response to the upward vertical movement of the transport membrane. A reservoir 78 at the end of the set 70 extends downwardly around the set 70 as a lower reservoir 146. It has been extended. A depression 136 is cast into the gel 42 and loaded with the substance to be analyzed. be done.

Variations in collection efficiency can be reduced by restricting the movement of the collection surface to a linear path and Collecting membranes within the collection area by minimizing tactile pressure and applying controlled tension in the plane of the membrane. can be minimized by stabilizing the Damage to the collection surface and reprinting removed and fractionated bands are accurately transferred.

The membrane is in the form of a sheet, giving it just enough size to hold the separated bands. It will be done. Create a border on this sheet with four edges, support it in a frame and connect it with two opposing edges. Clamp or secure the ends to the frame. The frame is moved relative to the plane of the gel by speed control means. The movement is restricted in the transverse plane by and transferred to this plane. The frame is moving away from the tension that creates line contact near and beyond the gel end. move. This provides a stable collection surface and allows the gel to be controlled below the damage threshold. the collection surface of the membrane under controlled pressure and under controlled tension.

In preferred embodiments to minimize the effects of surface contact, gel slabs The angle of membrane roll-up at the end of is physically maintained at or near zero. to create minimal contact pressure. During transport, this membrane is placed under controlled tension. put. A means of adjusting both of these necessary limits is the gel slab supporting the back side of the membrane. Contains a spring-loaded slidable block that operates within the surface. this membrane is tilted away from the membrane holding frame and towards the gel. This tilting effect and the gel retainer The movement of the membrane and the frame during transport creates tension in the membrane, which Relative to hold and position smoothly and reasonably relative to the end of the gel. The surface of the membrane close to its contact with the gel, which is oriented away from the direction of motion. Line contact or tightening across the gives a band. This pressure is the spring constant of the tilt spring is preselected as a function of . A slidable block allows the membrane to be removed in the process. Since the gel is in contact with the end of the gel holder, the membrane rolls up near the end of the gel. angle is kept to virtually zero, thereby maintaining contact pressure at a minimum level. ing.

A buffer reservoir in the chamber where the block slides holds the buffer that spills over on the back side of the membrane. do. Electrodes are placed in the solution, providing electrical continuity between the buffer solution and the membrane. It is being Preferably, the other (introductory or cathodic) end of the separation gel preferably includes a buffer reservoir as part of the gel holder or cassette. Ru. An electrode contacts this liquid, preferably moving the electrode into and out of the contact area. It is mounted on a component that vibrates outward.

The separation and transfer devices described above are most suitable for automated operation.

Separation and transport are restricted to modules arranged for this purpose and the membrane frame is moved vertically from this separation and transfer module to the traveling machine, and the traveling machine This is carried to the next processing module and finally to the detection module, where all modules is under the control of computer means, which also performs the identification of the separated fractions. Preferred embodiments are disclosed.

Referring now to FIG. 3, which is a cross-section of FIG. 2 taken along line 3--3, the module 34 can be seen in more detail. The cavity 150 has a bottom 48 and an open top 50. It is a four-sided container 46 having a side surface of 52.52° and two end faces of 54.54°. It is thought that it has a lower reservoir 1146 that spans the entire width between 52.52° on both sides. There is. The container 46 has a gel retainer 44 removably attached to one side of the lower storage tank 146. is attached. It was noted that the container 46 is narrower at this end than the other end. (See Figure 2). The gel cassette 44 is a first component (or extension reservoir) 60 It has an "I7" shaped cavity 58 having a gel 42 in its first component. is cast and the formed slab is aligned with its open end and at the other end. It is aligned with the wall 36 of the second component forming a liquid reservoir 62 for the buffer solution. slot a comb 64 for holding a combing tool, not shown in FIGS. 2 and 3; A depression 136 is formed in the gel 42. in the first component 60, preferably A slab of gel 42 approximately 1.5 nu++ thick forms the separation region, which will be discussed in more detail later. It is cast in alignment with the distal end of the first component 60, as discussed in detail. gel The retainer 44 is securely held by screws 66, 68 (FIG. 2).

A slidable set 70 is fitted into the wider portion of the container 46. this The set of parts forms a reservoir 78 which is filled with membrane-buffer solution 80 during use. The buffer solution then contacts the back side of the membrane 30 to form a liquid interface 82 . The electrode 88 It extends through the tank 78. One or more springs 84 are preferably fixed. The slideable set 70 is attached to the end of the slideable block 72 and the gel cassette. The membrane 30 in the component 60 is tilted towards the sandwich membrane 30 between the The front surface of the gel 42 is brought close to the end of the gel 42, so that the angle of roll-up is substantially zero.

Frame 28 bridges the ends of set 70 to expose membrane 30 to pinch line contact. It has the same dimensions.

Referring now to Figure 38, an example using only a limited amount of membrane-buffer is shown. Can be seen. This variant is almost identical to what has been outlined so far in this specification. Use one device.

The wider part of the container 46 is fitted with a slidable set 70°. child In this embodiment, the set 70" has a bottom back and a front by means not shown. from a block 72° with two sides to which the wall 74 is removably tightened; an open-top unit constructed of blocks 7 and 7, whose walls hold cores 76 in slots 75; 2 and beyond the wall 74 at least in the direction of the gel 42. It extends. A preferred material for constructing the core is Porex technology (Porex T Hydrophilic Poresox (1’c) REX, a product of ect+noLogies) l+), but this core does not contaminate the buffer and has suitable porosity and electrical properties. can be made from any porous material, such as felt or paper, that has I can do it. This portion of cents forms a reservoir t1178, and this reservoir, when in use, Flowing through the wick 76 in a controlled manner to form a liquid interface 82 against the back side of the membrane 30. It is filled with III solution 80. Depending on the distance of the core 76 from the membrane 30, the interface can be film-like or bead-like. Electrode 88 extends through reservoir 78 There is. One or more springs 84, which are preferably fixed, are attached to a slideable spring 84. 70' between the end of the slidable wall 70° and the end of the gel retainer 44. Tilt the front surface of the membrane 30 toward the sandwich membrane 30 between the The roll-up brings it close to the end of the gel in component 60. Frame 28 is a block The dimensions are such that the 72° ends are bridged to expose the membrane 30 to the contact. wick supply section can be replaced by a slot/throat supply section. This can be seen in Figure 4B. will be discussed below.

Turning now to FIG. 4 and overviewing the transfer region of the preferred embodiment, gel 42 is It is cast into set 44 so that the end of the gel is aligned with the end of the gel cassette. gel The upper part 44' of the cassette 44 is at a small vertical distance from the lower part. Gap 8 is extended and then cut to help limit surface contact on membrane 30 6 left. The end of the gel 42 is restricted to a tapered shape by the configuration of the component 60. Please note that This is the electrical focus of the separated band, i.e. It gives concentration. The end of the gel cassette 44 is connected to the end of the slidable cent 70. This makes the placement within the transfer area less critical. this is Also, the membrane 3 is connected between the respective ends of the gel cassette 44 and the slidable set 70. 0 to give a contact line, and as the frame 28 moves upward, as above. This creates tension on the surface of the membrane. Membrane-buffer 80 also overflows reservoir 78 as described above. are doing.

Referring now to Figure 4A, it is desirable to minimize the amount of membrane-buffer used. An overview of the transport area of a new example. In general, the description of the spilled set is also here. The same holds true. The sliding set 70゛ is slotted underneath as seen earlier. converting the net 75 into a slidable reservoir with a front wall 74 closed with a wick 76 be done. The wick 76 may touch the back side of the membrane 30, but is preferably shown in the figure. So far away.

In FIG. 4B, a simpler configuration is shown without the core 76. height Suitably reduced submerged slots 75 meter in buffer at a reasonable rate However, it has been found that the desired electrophoresis can be achieved in many cases.

In both of these latter two limited supply systems, the limited supply of buffer An overflow arrangement is not preferred as it also increases the probability of foaming due to lack of interface. There isn't. This creates distorted spots. If the interface 82 is too small, the surface tension It can become negative and contaminate the spots.

The frame 28 seen in Figure 5 is preferably made of plastic such as polystyrene. It is a thin plate with an opening 90 that fits into the membrane 30. Membrane 30 is normally Manufactured from iron, but only at the top and bottom, using adhesive and is bonded to the frame 28 by heat sealing or ultrasonic sealing. This drips onto the surface. Prevents any wave folds when directly applied loads. For the same reason, in fig. Cut the membrane slightly smaller than the opening 90 transversely to the direction of travel seen during transport. Although it is possible to cut it, it is preferable to seal the periphery during subsequent processing. Then, there are advantages to having overlapping membranes on all sides of the frame. . Therefore, it is possible to use either configuration depending on the particular system chosen. be. Box 28 preferably includes an identification area 92 in which barcode 94 is inserted. A sample identification section like this can be installed. Preferably, the frame 28 also has a reasonable machining arrangement. with orientation slots 28a to facilitate rowing and/or for signal positioning; do.

FIG. 7 shows a mechanism for moving the membrane frame 28 and the accompanying membrane 30 inside the base 12. A specific example of the transfer device 10 of FIG. 1 is shown in FIG. The specific example in FIG. in dynamic bench operation and therefore not integrated into an automatic handling system. It is also characterized by being targeted.

For example, the removable lid 401 is placed on the base 12 using the handle 402. . The base 12 is equipped with leveling means, e.g. adjustable feet 403, with leveling fingers. It may also have an indicator 404. Belt 405 connects pulleys 406 and 407. are connected to each other.

The membrane movement mechanism is shown in more detail in FIG. Pulley 407 is motor 4 It is placed on the shaft 408 of 09. The pulley 406 of the shaft 410 is A tire (not shown) is placed on the Dee 411, and the other end is covered with an elastic material. ). This tire rides on the vertical groove 412 of the body 411. Therefore, one edge 11) is in contact with the surface of the membrane frame 28. Compatible with tires On the other hand, there is a roller 413. In an alternative embodiment, roller 413 is removed and the tire presses the frame 28 against the opposite side of the groove 412 to obtain traction force. . In this case, in order to provide an appropriate coefficient of friction, the outside of the body 411, but The groove 412 is made of a low-friction technical plastic, such as Delrin. ’) (E.I. du Pont de Niemers (E, 1. du Pan) t de Nemours and Company, Wilmington, (commercially available from DE).

The detector device 414 is actually two combined optical detectors and an integrated light source. Forming part of the control system, the entry of the membrane frame 28 into the lower part of the mechanism and its stop in the operating position To confirm its presence in full engagement with the tool 415 and to ensure proper orientation of the frame. used in This is done by means of the positioning hatch 28a. That is, the frame When the bottom of 28 is fully engaged with the above-mentioned stop, the notch 28a The upper corner of the frame 28 is at the lower detector position that does not block the Blocks the light rays of the vessel.

During use, the base 12 (of FIG. 1) is attached to the platform 14 (this The system incorporates a leveling tool into the base 12 itself, as seen at 403 in FIG. (can be omitted). Usually the gel 42 (publicly available) is cast in the correct position. Weir stopper necessary for gel casting as well as the above-mentioned combing tool or depression forming tool (remove) and tighten the gel cassette 44 in its proper position in the container 46. do. As mentioned above, the inner membrane 30 in the frame 28 has a deep depression 1 connected to the carrier 26. A set of recesses 70 which are retained in the container 46 and are slidable are located at the large end of the container 46. It is installed in

The arm 140 was vibrated to place the electrode 38 on top of the gel 42 of the component 62. Buffer solution 80 is contacted and buffer solution 80 is placed in reservoir 78 .

The device of FIG. 7 has an electrode (not shown) installed on the underside of the lid 401. Ru. Therefore, the operator simply closes the lid and places the electrode in its proper position. Ru.

The recess 136 is loaded with the sample to be analyzed, and the electrode is moved so that the ions move toward the end of the membrane. Electrophoresis is performed as known by applying an appropriate voltage drop across 38 and 88. After a sufficient period of time has elapsed, the motor 18 is activated to drive the membrane 30 upward. The separated parasitic material is recovered and collected in a known manner.

Then, the membrane frame 28 is manually or automatically removed from the membrane carrier 26, and the membrane 30 and and the separated band of material thereon can be directed to subsequent processing.

Previous descriptions have focused on the relationship between horizontally arranged gel slabs and vertically movable membranes. However, the exact aspects of the method and apparatus are such that the membrane has at least a transport area. It can also be applied well to vertical gels in devices that are movable horizontally in the area. So Additionally, other arrangements of membranes and gels are possible, as will be readily appreciated by those skilled in the art. Ru. For example, the membrane is moved 45 degrees relative to the gel as the buffer solution rises. It is also possible. Therefore, the description herein refers to substantially perpendicular contact and membrane and gel configurations, although other configurations are preferred by researchers herein. considered suitable.

Figure 6 shows a device generally designated 600 that can be incorporated into a The preferred shape of the device 1o for automatic processing in-system is shown.

Details of the base 12 are not shown. This detail is generally shown in Figure 8 above. The following devices are used. Initially, the frame 28 is manually removed and the frame 28 is removed as shown in FIG. It is inserted into engagement with stop 415 which is placed in the operating position. Start electrophoresis Once the frame has been moved, the motor 409 is activated to drive the frame upward at a controlled speed. Collect the separated band of DNA.

When the electrical operation is completed, insert the frame 28 through the slot 416 in the cover 401. and formed between the strip body 601 and the body 602 of the traveling machine 603. Drive into groove 615. This is a motor controlled by a motor not shown. The ear 604 is clamped to one of the strips 601. Traveling machine 6 03 is moved on the guide rail 605 in a suitable linear bearing (not shown). move. This rail is attached to structure 606. Located above the traveling machine 603 Detector 614 operates in two ways. The frame 28 is sent to the top of the groove 615. , the detector 615 detects the presence of the frame 28 and the absence of the top edge. This causes the control system to stop the drive system. The drive system is at the top of the frame 28 located alongside the detector. The frame 28 is fed to the bottom of the groove 615 and the drive system is placed on top. Once activated, detector 614 detects the absence of frame 28 and the presence of the top edge. sense and stop the drive system again to obtain a reasonable position.

The traveling machine 603 has a heald 609 (preferably a timing control) fastened to the traveling machine 603. a pair of pulleys 608 (only one of which is visible in the figure) operating on a is moved by a motor 607 that drives one of the two.

During the process with the device 10 for separation and transfer, at least one It is equipped with a vertical transport device similar to that described for module 10, but Others are processing modules 200 that differ in their internal elements. This kind of module The tool simply uses the appropriate reagents and includes drying means if necessary to separate the DNA fractions. Can be equipped to animate and transform. Then the subsequent module No mixing, washing, blocking, heating and chemiluminescent labeling is required. Labeling with quality can be done in the pond module before labeling. Frame 28 can be used as needed. may be moved from module to module as needed. One side fixes the frame 28. between the cellar 1- of the vertical transport system, one in the fixed module and the other in the traveling machine 603 The steps passed to the module are sufficient to clean all intervening modules or parts thereof. Raising the bottom of the frame to a height facilitates this processing step. did Therefore, this aspect supports irregular machining processes with any number of intermediate stations. hold Module 200 can also perform all of the processing steps described above or a single module. It is also suitable for carrying out different machining stages in the mill.

After the membrane 30 has been completely processed, it is transferred to the detection module 300 where the membrane is 30 in position for reading by a detection device 500, preferably a CCD camera. put. The data from the detection device 500 is sent to the process control means 400, preferably a mini calculation. The sample is then fed to a machine where the band pattern is analyzed by methods known in the art.

Once the information on membrane 30 has been read or scanned, it can be disposed of for reasonable disposal. discharged together with the frame 28 carrying the membrane into a sanitary waste section (not shown), or Make it “fall”. The used property is removed from the module 300 using gravity and an escape port. Release through the bottom slot (not shown) and the table 14 to allow the fall. i’li! and the necessary light shielding and location. 11711 method is preferred stomach.

This processing cycle is of course repeated after the drop. Set 70 is load operation If not pulled back in, the frame 28 would be lifted onto the top edge of the set 70. That is clear. Referring now to FIG. 9, the independent, semi-autonomous configuration in the configuration of FIG. Configuration of the device 10 showing means for this type of pullback in modules intended for dynamic operation You can see a cross section of the part. When the lid 401 is raised, the tightening thread 416 is attached to one end. Since it is connected to a tightening tool 418 fixed to the lid 401, no tension is applied to this. Karu. Tension thread 416 is a nylon filament or the like. This tightening thread is The set 70 (membrane tension act as a pressure barrier against the force). Negative tension on the tension thread 416 The load pulls the slidable set 70 out of contact with the cassette 44 compressing the spring 84. return. The opposite effect occurs when the lid is closed. Lid that swivels around a pivot axis In automatic operation, which does not include Tension is applied by a noid or the like, or more preferably, it is connected to the traveling machine 603. When used in conjunction with a lever activated by the presence of a traveling machine, the correct position can be maintained. The cam can be driven around the position.

Module 300 is identical to that used in modules 10 and 200. Well equipped with vertical transport mechanism, running above the used frame/membrane 28/30 transport to the machine 603, which further extends the extension structure 6 on the 4 sides of the table 14. 06 (and each attached element), and a fall can occur there. This will also be clear to those skilled in the art.

Regular and reasonably timed machining allows the creation of various stepper motors. It is under the control of a control means 400 which causes it to move and perform the necessary functions. time balance To obtain this, it is possible to duplicate the module and process two or more membrane frames 28 at the same time. can. Similarly, if the cleaning step is also achieved in duplicate modules, the membrane frame 28 can be returned towards the starting end of the transport system of this type of module.

Optionally, if the protocol may require temperature control for the gel 42 , temperature control means with attached temperature sensing means and controller (not shown) 43 (shown in FIG. 4). This is located under the gel cassette 44. There is. For heating, a Minco enclosed immersion heater (HK5418R5) was used. 1,8L12B-9102 type) and an RTD as a detector.

We have determined that the unbound leading edge of membrane 30 traverses the surface of gel 42 under load. It has been found that the edges of the gel 42 can be damaged when passing through. this The leading edge is the first edge of membrane 30 upon entry or exit of membrane/membrane set 30/28. This is the part that passes near the gel. The edges around the outside of the join seam are left unbound. This is the joining edge. This edge shreds the gel during loading and unloading operations, or have a tendency to damage. Therefore, in the above specific example, A device was equipped to unload the contact pressure during such loading (see, for example, FIG. 9).

The inventors also found that a well-manufactured frame/membrane set 28/30 has a tension in the membrane 30. It was also found that due to the force, the frame always curved slightly away from the membrane. (child The curvature of is exaggerated in FIG. 10 for illustrative purposes). This means that in this case This is particularly true regarding the preferred ultrasonic coupled units.

Assembly uses ultrasound to bond one end of the membrane to the frame. Then remove the frame from the membrane. the concave surface thus formed faces the membrane. Next Then, the opposite edge of the frame membrane is ultrasonically bonded to the frame without applying some tension to the membrane. Ru. The tension required is sufficient to keep the membrane straight and flat. This is it This can be achieved, for example, by the experimenter pulling on the free end of the membrane or by other means. I can do it.

Other methods of manufacturing tensioned frames include: ultrasonic bonding a vacuum fixture having a vacuum pad under the anvil of the agent and a pad under the window area of the frame; Place the frame on top. Place the membrane in place; engage both pads. binder and fix one end of the membrane to the frame. Stop the vacuum and invert the frame in the fixture. Ru. Operate the vacuum only below the anvil and manually clamp the frame on both sides near the center. sample. Manually slide the frame under the clamp and pull it toward the operator. over a fixed object bending the edge the rator is pulling away from the framed membrane. to rise to. A binding agent is applied to secure the second end of the membrane to the frame. stop vacuum Then, loosen the clamp and allow the spring to return the frame to its very slightly curved shape.

Using this curvature, we also make progress in other specific examples, as seen below. Obtained.

In Figure 10, the sliding block and associated springs eliminate the means for direct pressure relief. Simplified embodiment that avoids contamination of the gel during loading even if no means are included An example is shown. First, force the frame 28 to slide at a constant angle to the vertical. . An angle of about 10 degrees is preferred. Component part 414 is vertical, and part 415 is vertical. Approximately 0. D45 inches, that is, a distance slightly smaller than the thickness of the frame . Moving the components 414 to larger spacings may cause unnecessary frame bending. It will be. The preferred angle of the first part of the guiding groove is about 10 degrees, but there are important The principle is to clean the end of the gel and contact the far wall at an angle that does not fit. . During loading, the frame 28 and its associated membrane 30 form a groove 412 at the outer edge of the frame 28. is driven by a rubber tire roller 500 acting within the wall 1 Suppressed by 00.

A system having pairs of rollers on both sides and driving all rollers is preferred. Low The 500 is covered with durometer rubber 70 to provide approximately 12 mils of twist. is placed on frame 28. By approaching at this angle, the lower end of the frame 28 is attached to the gel 4 When the frame 28 hits the far wall 100 cam-driven towards the The mating edges allow cleaning the ends of the gel 42. In continuous motion, the frame is When entering, it fits into the vertical component 413 of the groove 412'. This wall is gel 42 (and carrier 44) by the thickness of frame 28. of the present invention In a preferred embodiment, the frame thickness is 0.060 inches and 0.036 inches thick. A spacing of ±0001 inches provides a gap of 0003 inches. Of course this The dimensions of these will vary depending on the overall dimensions of the septum and also depending on the desired required dimensions. It can change. Continuous motion bends the frame and the vertical portion of the groove 412° as the frame advances. Conforms to minute 413. The groove 412° is a structure 420 provided on the opposite side of the unit 10. formed inside. Next, the l1lI component part 414 with the sloped top of the frame 28 is Clean the distal end and attach it to the far side of the expanded section 415 towards the starting position. It assumes a vertical position as it is driven downward.

Here, the position of the groove, the contact of the roller, and the slight curvature away from the frame membrane mentioned above will be explained. The bend causes the membrane 30 to slide under tension through the end of the gel carrier 44, causing the frame 28 to be driven upwardly. cause the desired line of tension to occur beyond contact with the end of the gel 42 when moved. Ru. Continuous upward movement of the frame 28 towards the extension 415 leaves the top back for evacuation. Cam drive the sloped component 414.

This configuration allows for considerable size reduction and reduces the number of parts.

FIG.1 FIG.2 FIG.3 FIG, 3A FIG.4 FIG. 4A FIG. 4B FIG.9 International search report PCT/us 92/+0281 front page continuation (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, S E), AU, BR, CA, JP, RU, UA (72) Inventor: Bartson, Charles William.

junior 197320 Tsukland B-O Box 154 for USA Praware

Claims (1)

[Claims] 1. (a) an electrophoresis module containing a gel containing a solution with molecular fractions; (b) means for performing electrophoresis along the periphery of the flexible membrane; an electrophoretic transport membrane comprising a rigid frame supporting a membrane; (c) the relative position of said electrophoretic transport membrane to said electrophoretic module; The above molecular fraction is transferred from the above gel to the above flexible membrane by moving it in close proximity to this. and (d) means for receiving said electrophoretic transport membrane into said electrophoretic module; a means for tensioning said flexible membrane as it moves proximate to the molten membrane; A device for gel electrophoresis that electrophoretically separates molecular fractions in a liquid using a gel. 2. In the above electrophoresis module (a), means for performing the above electrophoresis (i) a cassette having a first reservoir adapted to receive a buffer solution and a first electrode; , and an extension adapted to receive a gel, wherein the first reservoir is connected to the extension. (ii) a buffer solution and a first a second reservoir having two electrodes and oriented in a second position relative to said extension; A gel characterized by comprising a bath and further containing a solution having a molecular fraction. be located within the expanded portion and between the first storage tank and the second storage tank; It is also characterized by applying a potential difference between the first electrode and the second electrode. Direct the molecular fraction to the end of the gel in close proximity to the electrophoretic transport membrane (b) using Claim 1, further comprising the step of electrophoretically moving and separating. Device. 3. In the electrophoresis module (a), the cassette is 1 and said extension are placed substantially perpendicular to each other; The expansion portion extends horizontally in the lengthwise direction, and the first reservoir extends in the lengthwise direction. A claim characterized in that it is formed in an "L" shape extending vertically. 2. The device according to 2. 4. The above extension has a width at least 10 times greater than its height, and a flat surface therein. A claim characterized in that it has a rectangular cross section suitable for casting gel slabs. The device according to scope 2. 5. The above extension is received to contain the solution containing the molecular fraction for electrophoresis. an upper surface having an opening therein suitable for casting the requisite recess in the gel; 3. The device according to claim 2, characterized in that: 6. the extension has a distal end, at least the distal end being connected to the first distal end; It is formed to have a height that gradually decreases from the storage tank and extends along this height. 3. The device according to claim 2, characterized in that: 7. a vibrable, apertureable device in which the first electrode is connected to an electrophoresis module; 3. Device according to claim 2, characterized in that it is arranged in sections. 8. The buffer in said second reservoir (ii) is electrophoretically transferred to said reservoir (ii). of said transfer membrane (b) via a slot formed in one end adjacent to membrane (b). 3. Device according to claim 2, characterized in that it is in liquid contact with the flexible membrane. 9. Said slot allows for the transfer of buffer from said reservoir (ii) to said transfer membrane (b). Claim 8, characterized in that it contains therein a wick for measuring the flow rate. equipment. 10. Said means (c) for moving said electrophoretic transport membrane is said rigid body. including a rotating component in contact with the frame, the electrophoretic transport membrane a motor sufficient to move it relative to and in close proximity to module (a); in operative connection with the motor and the drive coupling. Apparatus according to scope 1. 11. The means (d) above for imparting tension to the flexible membrane is applied at the desired tension. said flexible end adjacent said electrophoretic transfer membrane (b) of said gel. A claim characterized in that it includes means for creating line contact with a flexible membrane. The device according to scope 2. 12. Said means for producing line contact are placed in said second reservoir (ii). the removable components and tilting means; The element is characterized in that the flexible membrane is brought into contact with the gel and separated. 12. The apparatus according to claim 11. 13. In the above electrophoretic transport membrane (b), the above rigid frame is the above flexible membrane. curved away from each other, the flexible membrane having one or more non-bonding edges; further comprising: Means (c) further comprises arranging said rigid frame in such a way that the unbound edges do not contact said gel. placed to drive through a slot located at a sufficient distance from the vertical plane. Molecular fractionation is carried out by driving at least two rollers and the above-mentioned rigid frame to a vertical position with a cam. from the gel onto the flexible membrane; ; furthermore, said means (d) for applying tension to said flexible membrane is said roller; Also includes a combination of the above-mentioned curved and horizontal rigid frame and the above-mentioned second storage tank. 3. The device according to claim 2, further characterized in that: 14. Note that the above slot is oriented approximately 10 degrees from the vertical direction. 14. The apparatus according to claim 13, wherein the apparatus comprises: 15. (1) (a) Electrophoresis module containing a gel containing a solution with molecular fractions (b) along the periphery of the flexible membrane; an electrophoretic transfer membrane including a rigid frame supporting the membrane; (c) the relative position of said electrophoretic transport membrane to said electrophoretic module; The above molecular fraction is transferred from the above gel to the above flexible membrane by moving it in close proximity to this. and (d) means for receiving said electrophoretic transport membrane into said electrophoretic module; separation comprising means for tensioning said flexible membrane as it moves proximate to the roll; and a direct wiping transfer module; (2) a separation and direct membrane transfer module as described above; one or more membrane processing modules placed in a linear arrangement with the module (1); 3) The above separation and direct wiping transfer module (1) and membrane processing module (2) and detection modules arranged linearly; (4) the above modules (1); ), (2) and (3) operable to move the above electrophoretic transfer membrane in and (5) modules (1), (2) and (3) above; a control operable to aggregate and control the functions of the linear transport means as described above; Direct wiping transfer of molecular components to electrophoretic transfer membranes, including control means, Automatic gel electrophoresis equipment consisting of processing and detection of components therein. 16. (a) A gel containing a solution with a molecular fraction in it, along which an electrical current is introduced into an electrophoresis module having means for performing pneumophoresis; (b) an electrophoretic transfer membrane comprising a rigid frame supporting a flexible membrane along its periphery; (c) positioning the electrophoretic transport membrane in contact with the gel; Relative to and close to the pneumophoresis module, move the molecular fractions up. From the above gel to the above flexible membrane: (d) when said electrophoretic transfer membrane moves in close proximity to said electrophoretic module; A method for gel electrophoresis of molecular fractions, which involves the steps of applying tension to the above-mentioned flexible membrane. Law.